Project Summary For modern flow cytometry, high-throughput multiplexing is extremely important because of the great need in analyzing a large number of biomolecules on and in a single cell. The multiplexing performance of flow cytometry is hindered by the probe performance including their fluorescence brightness and emission bandwidth. However, there is renewed interest and effort to develop highly multiplexed (e.g. ~20-50 probes) fluorescence based flow cytometry analysis of cells. For this high degree of multiplexing with fluorescence, both probe brightness and narrow emission are critical - specifically, because of how compensation for spectral spillover works, a very bright probe greatly facilitates multiplexing by minimizing spillover spreading, which is a key driver for extremely bright probes in flow cytometry. The ability to detect low- expression antigens is also another important capability provided by very bright probes. Recently, we and others have developed fluorescent nanoparticles based on semiconducting polymers called Pdots. The motivation of adapting fluorescent semiconducting polymers into nanoparticle labels stems from a number of favorable characteristics, such as large absorptivity, high quantum yield, fast emission rates, and excellent photostability. The resulting Pdots exhibit extraordinarily high fluorescence brightness under both one-photon and two-photon excitations, a factor of 102 - 104 higher than conventional fluorescent dyes, and a factor of 10-103 higher than Qdots depending on the particle size. We have successfully designed and prepared a Pdot series with 405nm excitation. Following on this successful development, we propose to design three additional Pdot series for flow cytometry, excitable at 355nm, 488nm, and 561nm, thus matching the laser wavelengths of existing flow cytometers.